Abstract
The Cngb1 locus-encoded β-subunit of rod cGMP-gated cation channel and associated glutamic acid rich proteins (GARPs) are required for phototransduction, disk morphogenesis, and rod structural integrity. To probe individual protein structure/function of the GARPs, we have characterized several transgenic mouse lines selectively restoring GARPs on a Cngb1 knockout (X1−/−) mouse background. Optical coherence tomography (OCT), light and transmission electron microscopy (TEM), and electroretinography (ERG) were used to analyze 6 genotypes including WT at three and ten weeks postnatal. Comparison of aligned histology/OCT images demonstrated that GARP2 accelerates the rate of degeneration. ERG results are consistent with the structural analyses showing the greatest attenuation of function when GARP2 is present. Even 100-fold or more overexpression of GARP1 could not accelerate degeneration as rapidly as GARP2, and when co-expressed GARP1 attenuated the structural and functional deficits elicited by GARP2. These results indicate that the GARPs are not fully interchangeable and thus, likely have separate and distinct functions in the photoreceptor. We also present a uniform murine OCT layer naming nomenclature system that is consistent with human retina layer designations to standardize murine OCT, which will facilitate data evaluation across different laboratories.
Highlights
Phototransduction, the process by which light stimuli is captured and converted into an electrical response, is initiated in rod and cone photoreceptors of the retina[1]
Murine GARP1 is a 550 amino acid protein that is four-fold less abundant than the β-subunit, while GARP2 is a 326 amino acid protein that is five-fold more abundant than the β-subunit
Connections between the plasma and disk membranes have been reported in amphibian, rodent, and bovine rod outer segments based on freeze-fracture electron microscopy[22,23], and cryo-electron tomography[15,24] analyses
Summary
Phototransduction, the process by which light stimuli is captured and converted into an electrical response, is initiated in rod and cone photoreceptors of the retina[1]. Within the rod outer segment disks, a photon of light activates the visual pigment, 11-cis-retinal, causing a change in conformation from 11-cis to all-trans This leads to activation of rhodopsin and subsequently transducin, an intracellular messenger, which activates cGMP phosphodiesterase 6 (PDE6) via displacement of its inhibitory γ-subunits. Evidence suggests that soluble GARP2 interacts with peripherin-2 in the rim region of the photoreceptor disk[20] In addition to their functional role, the GARP region on the β-subunit forms a physical link between the plasma and disk membranes[15,20,21]. Knockout of the β-subunit and GARPs14 (X1−/−) results in loss of structure and function, including disruption of disk/plasma membrane interactions[15] and a > 90% decrease in CNG α-subunit levels[14]. We present a system for murine optical coherence tomography (OCT) layer designation, that conforms with recently published human OCT guidelines[25] and provides the basis for a standardized noninvasive assessment of murine posterior segment structure
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